应用于结核病治疗的分枝杆菌噬菌体D29的免疫原性研究

Research of immunogenicity of mycobacteriophage D29 toward tuberculosis therapy

目的观察分枝杆菌噬菌体D29采用不同给药途径在豚鼠模型中的免疫原性，为噬菌体应用于结核病治疗奠定基础。方法Hartley豚鼠分别以吸入、滴鼻、皮下注射3种途径分组，在35d内给予分枝杆菌噬菌体D296次，设7H9培养基溶剂为对照组和0．85％氯化钠溶液为阴性对照组，通过噬斑减少中和实验（PRNT）检测每周豚鼠血清的中和抗体效价，通过ELISA测定第35天血清中IL-2、IL4、IFN-7水平。统计学处理采用方差分析和非参数检验。结果两对照组均未测出中和抗体；吸入组和滴鼻组仅在第7、14天检出-过性低效价中和抗体（〈1：100）；皮下注射组持续产生中和抗体，第35天效价高达1：16365．6。给予分枝杆菌噬菌体D2935d后，各实验组及对照组豚鼠血清中IL-2（X^2-2．7605，P〉0．05）、IL-4（F-2．17，P〉0．05）、IFN-γ（F=0．75，P〉0．05）浓度均差异无统计学意义。结论吸人、滴鼻给药产生的抗噬菌体D29中和抗体效价明显低于皮下注射给药；噬菌体D29未引起细胞因子浓度的变化，提示不会破坏机体Thl／Th2免疫平衡。

Objective To investigate the immunogenicity of mycobacteriophage D29 （phage D 29） in guinea pig models with different delivery routes, and provide information for the application of phages in tuberculosis （TB） therapy. Methods Harttey guinea pigs were administrated with phage D29 through inhalation, intranasal drop or subcutaneous injection for 6 times within 35 days. 7H9 broth aerosol inhalation and 0.85% NaC1 solution aerosol inhalation were set as solvent and negative controls, respectively. Anti-phage D29 neutralizing antibodies in sera collected weekly were measuredby phage reduction neutralizing test （PRNT） and cytokine levels （interleukin-2, interleukin-4 and interferon-）＇） were detected at day 35 by enzyme linked immunosorbent assay （ELISA）. The data wer~ analyzed by ANOVA and nonparametric test. Results Neutralizing antibodies were both negative in two control groups, while low-titer neutralizing antibodies （below 1：100） appeared in inhalation and intranasal drop groups only at day 7 and day 14. Nevertheless, neutralizing antibodies were continuously detected in subcutaneous injection group, which increased rapidly and reached 1 ： 16 365.6 at day 35. After 35 days of experiments, serum concentrations of interleukin-2 （X^2= 2. 7605, P〉 0.05）, interleukin-4 （F=2.17,P〉0. 05） and interferon-γ（F=0.75,P〉0. 05） among three treatment groups and two control groups were all not significantly different. Conclusions The titer ofanti-phage 29 neutralizing antibodies induced by inhalation or intranasal drop administration of phage D29 are both significantly lower than subcutaneous injection. Phage D29 administration doesn＇t change the levels of cytokines, which indicates that it may not break the helper T cell （Tb）1/Th2 balance.